We have recently demonstrated that Methotrexate (MTX) in doses which inhibit pyrimidine and pyrine synthesis results in a 5 fold intracellular accumulation of 5-Fluorouracil (5-FU) in L1210 cells. This enhanced intracellular accumulation of 5-FU is dependent on an increased availability of phosphoribosylpyrophosphate(PRPP), presumably the result of purine synthesis inhibition, the consequence of tetrahydrofolate depletion that occurs following inhibition of dihydrofolate reductase (DHFR) by MTX. This sequence of MTX preceding 5-FU leads to synergistic killing of these cells in the soft agar cloning system. We wish to evaluate the biochemical consequences of this sequence in other cell lines, specifically colon and breast cancer cells to better determine the possible clinical efficacy of this sequence. The therapeutic effectiveness of this sequence will be tested in mice bearing various tumors. In addition, the 5-FU tissue distribution in mice will be determined following MTX to find sites which may be preferentially affected by this sequence. The 5-FU derivatives which accumulate within the cells have been determined by high pressure liquid chromatography methods to be FUMP, FUDPG, FUDP, and FdUMP. There was also a 3 fold increase of FUTP into RNA. Although these interactions are dependent on increased quantities of PRPP from purine synthesis inhibition, the actual phosphorylation of 5-FU occurs by pyrimidine PRPP transfeerase, the orotidylate decarboxylase-OMP synthetase enzyme complex. Pyrazofurin (PF) which inhibits the activity of this enzyme complex will prevent the MTX-5-FU interaction. We wish to isolate this complex and study the kinetics of these interactions. This may have clinical relevance since several drugs such as allopurinol, a drug used frequently in cancer patients, also inhibits the activity of this enzyme complex. Other agents which inhibit purine synthesis and result in increased PRPP levels also induce an enhanced intracellular 5-FU accumulation, and inhibitors of PRPP synthetase prevent both the PRPP increase and the enhanced 5-FU accumulation that follows MTX. We wish to evaluate the biochemical interactions of these drugs and their subsequent antitumor activity in more detail to better design rational sequential drug therapies of cancer.